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Endocrinology Vol. 138, No. 4 1621-1626
Copyright © 1997 by The Endocrine Society

ARTICLES

Endotoxin Decreases Corticotropin-Releasing Factor Receptor 1 Messenger Ribonucleic Acid Levels in the Rat Pituitary1

Jean-Michel Aubry, Andrew V. Turnbull, Giacomo Pozzoli, Catherine Rivier2 and Wylie Vale3

Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037

Address all correspondence and requests for reprints to: Dr. W. W. Vale, The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bacterial endotoxins produce profound activation of the hypothalamo-pituitary-adrenal axis, mediated by stimulation of hypothalamic CRF neurons. Although a number of studies have described direct pituitary actions of inflammatory mediators, the effects of inflammatory stimuli on the sensitivity of corticotropes to CRF remain to be elucidated. The aim of this study was to determine the effects of inflammatory stress on the CRF receptor 1 (CRF-R1) messenger RNA (mRNA) levels in the rat pituitary.

The systemic injection of endotoxin [lipopolysaccharide (LPS); 50 µg/kg, iv] increased plasma concentrations of ACTH and corticosterone. Ribonuclease protection analysis of total RNA isolated from individual whole pituitaries indicated that LPS produced a significant decrease in CRF-R1 mRNA that was evident by 2 h after injection (to 57% of control) and more marked by 6 h (to 38% of control).

To evaluate whether the decrease in CRF-R1 mRNA was dependent upon increased exposure to CRF and/or vasopressin (AVP), LPS was injected with an anti-CRF antiserum, a CRF receptor antagonist (Astressin), or anti-AVP antiserum. A strong inhibition of the ACTH response to LPS was produced by pretreatment with anti-CRF antiserum, Astressin, or anti-AVP antiserum. However, these treatments had no effect on the decrease in CRF-R1 mRNA produced by LPS, indicating that neither CRF nor AVP are obligatory mediators of this pituitary response.

The hypothesis that LPS might have direct pituitary effects on CRF-R1 mRNA levels was tested in vitro. Indeed, decreases in CRF-R1 mRNA to 43% and 53% of the control level were observed in rat anterior pituitary cell cultures that were treated with either LPS itself or the inflammatory mediator interleukin-1ß, respectively. Collectively, these results show that CRF receptor mRNA levels in the pituitary of the rat are markedly reduced by systemic LPS treatment and that this decrease is not dependent upon increased exposure of the pituitary to CRF or AVP, but may involve direct effects within the pituitary of either LPS itself or ensuing cytokine production.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE INTERACTION between the immune and neuroendocrine systems is now well recognized. Thus, inflammatory stress such as that observed after the injection of lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria which induces the synthesis and release of several immunoregulatory molecules, such as interleukin-1ß (IL-1ß), tumor necrosis factor-{alpha} (TNF{alpha}), and IL-6, elicits a number of neuroendocrine responses (see reviews in Refs. 1–3). Several studies using antisera or antibodies to cytokines or their receptors have also illustrated the importance of cytokines such as IL-1 in endotoxin induction of ACTH release. Thus, in response to LPS injected ip or iv, inhibition of IL-1 dramatically blunts ACTH secretion (4, 5). Accumulating evidence suggests that the response of the hypothalamo-pituitary-adrenal (HPA) axis to bacterial endotoxins is mediated by activation of hypothalamic CRF neurons. Indeed, numerous studies (6, 7, 8) have reported that LPS or cytokines such as IL-1 or IL-6 stimulate CRF gene expression in the hypothalamic paraventricular nucleus (PVN) and cause profound activation of the HPA axis (see reviews in Refs. 1 and 2). Supporting this concept, passive immunoneutralization of CRF inhibits the activation of the rat HPA axis by IL-1, IL-6, TNF{alpha}, or LPS (9, 10, 11, 12, 13).

CRF, the primary mediator of the activation of the HPA axis, is a 41-amino acid peptide synthesized in the parvicellular division of the PVN (14). The CRF neurons in the PVN project to the median eminence, where CRF is released into the hypophyseal portal circulation to stimulate ACTH secretion from the anterior pituitary (15). CRF exerts its biological effects by initially binding to membrane-bound receptors. Several subtypes of CRF receptors have recently been identified, and they are encoded by two different genes. They are members of the calcitonin/vasoactive intestinal polypeptide/GRF subfamily of receptors characterized by seven membrane-spanning domains. CRF-R1, the first CRF receptor subtype reported, was initially cloned from a human ACTH-secreting pituitary adenoma (16) and subsequently from AtT-20 cells (17) and human (17) and rat brain (18, 19). Binding of CRF to this receptor elicits activation of adenylate cyclase. In situ hybridization (20) and ribonuclease (RNase) protection analyses (16, 17, 18, 21) have revealed that the most abundant form of CRF receptors in the pituitary is CRF-R1. CRF-R1 message is detectable in the majority of anterior lobe corticotropes (20) and in intermediate lobe corticotropes.

The levels of pituitary CRF receptors are modulated during altered activity of the HPA axis, such as that after adrenalectomy or stress (22, 23). Furthermore, after systemic endotoxin administration, CRF-R1 transcription is strongly activated in the PVN and supraoptic nuclei of the hypothalamus (24). Given the importance of the HPA axis response to survival and recovery following endotoxemia (25, 26), we sought to determine the effects of systemic inflammation on pituitary CRF-R1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Experiments were carried out on adult male Sprague-Dawley rats (240–260 g BW) purchased from Harlan Sprague-Dawley Laboratories (Indianapolis, IN). They were maintained on a 12-h light, 12-h dark cycle (lights on at 0600 h) and provided rat chow and water ad libitum. All procedures described were approved by The Salk Institute animal care and use committee.

Rats were anesthetized with halothane (3% halothane and 97% oxygen) 48 h before experimentation and equipped with indwelling jugular venous catheters. The cannulas were constructed from a 3.5-in. length of SILASTIC brand catheter (inserted into the vein; Dow Corning, Midland, MI) connected to PE-50 tubing and filled with sterile heparinized saline. After insertion into the jugular vein, catheters were exteriorized at the nape of the neck to permit blood sampling and iv injection of drugs/antisera in conscious undisturbed animals. At the end of each experiment, animals were killed by decapitation, blood samples were collected, and individual pituitaries were dissected, frozen in liquid N2, and stored at -70 C until RNA extraction.

Reagents and treatments
LPS (Escherichia coli serotype O26:B6; code L3755, lot 20H4025, Sigma Chemical Co., St. Louis, MO) was dissolved in PBS and injected iv at a dose of 50 µg/kg. Sheep antirat/human CRF antiserum (code 253–228), normal sheep serum (control) and rabbit anti-AVP antiserum (code 277–94), or normal rabbit serum (control) were injected iv (0.15 ml/100 g BW) immediately before LPS treatment. The CRF receptor antagonist, Astressin, was synthesized using solid phase methodologies as previously described (27). Astressin (0.3 mg/kg in 0.25 ml 0.9% saline-0.1% BSA) was injected 0, 0.5, 1.5, 2.5, 3.5, 4.5, and 5.5 h after LPS treatment. Recombinant human IL-1ß was a gift from Dr. Tony Troutt (Immunex Corp., Seattle, WA) and was used in cultures of rat anterior pituitaries at a concentration of 1 nM, a dose shown to maximally stimulate pituitary cells (28).

Cell cultures: treatment
Anterior pituitaries from male Sprague-Dawley rats (180–200 g) were enzymatically dispersed by collagenase as previously described (29, 30). Primary cultures were established by plating the dispersed cells (5 x 106/6-cm dish) in tissue culture dishes in ß-PJ medium (29) supplemented with 2% FBS. The cells were incubated at 37 C in a humidified atmosphere consisting of 7.5% CO2 and 92.5% O2 for at least 3 days, and the experiments were performed within 5 days after cell dissociation. Before each experiment, the cells were rinsed three times with 3 ml ß-PJ medium supplemented with 0.1% FBS and allowed to equilibrate for 24 h. All treatments were performed in triplicate and repeated twice.

RNA extraction
Total RNA was extracted either from individual pituitaries in the in vivo experiments and from individual dishes of rat anterior pituitary cultures, using the RNeasy kit (Qiagen, Hilden, Germany). The average yield of RNA was 40–60 µg/individual rat pituitary from in vivo experiments and 35–40 µg/5 x 106 rat anterior pituitary cells in culture.

RNase protection assay
RNase protection analyses were performed as described (31, 32). Briefly, a 411-nucleotide riboprobe containing a 345-nucleotide antisense sequence specific to CRF-R1 receptor was generated using SP6 RNA polymerase in the presence of [{alpha}-32P]UTP (800 Ci/mmol) and the plasmid PMP-1 as template (18). Rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal loading control. A 244-nucleotide riboprobe resulting in a protected fragment of 134 nucleotides was synthesized with SP6 RNA polymerase and [{alpha}-32]UTP (800 Ci/mmol), using the rat GAPDH plasmid as template (pTRI, Ambion, Austin, TX).

RNase protection analyses were performed by hybridizing 20–25 µg total RNA in 24 µl deionized formamide plus 6 µl hybridization buffer containing 3.5 x 105 cpm CRF-R1 and 8000 cpm GAPDH riboprobes. After heating at 80 C for 5 min, the samples were hybridized at 45 C for 15 h and subsequently digested by RNase (200 µg/ml RNase A and 350 U/ml RNase T1) at room temperature for 60 min. The samples were resolved on 5% polyacrylamide-8 M urea gels. Quantitative analysis was performed using the PhosphorImager system (Molecular Dynamics, Sunnyvale, CA) and the ImageQuant 4.0 software package. The intensity of the protected CRF-R1 fragment was normalized to the intensity of the protected GAPDH fragment of the same sample, and results are expressed as corrected arbitrary units.

Total RNA extracted from individual rat pituitaries from each in vivo experiment were subjected to RNase protection analyses over two or three separate assays, and each in vivo study was performed twice. RNA extracted from individual dishes of cultured rat anterior pituitary cells were analyzed within a single assay, with each experiment performed a total of three times.

ACTH and corticosterone assays
Plasma ACTH concentrations were determined using a two-site immunoradiometric assay (Allegro, Nichols Institute, San Juan Capistrano, CA) as described previously (33). Assay sensitivity was 5 pg/ml, and coefficients of variation at a concentration of 330 pg/ml were 2.4% within an assay and 15.7% between assays, respectively.

Plasma corticosterone concentrations were determined in diluted plasma samples (diluted 1:8 in assay buffer, sodium phosphate EDTA azide-0.1% BSA), heated to 60 C for 45 min. Diluted samples were incubated overnight at room temperature with 125I-labeled corticosterone, normal rabbit serum and a rabbit anticorticosterone-3-BSA antiserum (final dilution, 1:30,000) obtained from Dr. G. Niswender (Colorado State University, Fort Collins, CO). Precipitation was accomplished by a 30-min incubation with sheep antirabbit IgG antiserum followed by centrifugation.

Statistical analysis
All data are presented as the mean ± SEM. Results were analyzed by ANOVA, followed by Dunnett’s or Bonferroni’s test where appropriate, for comparison between group means. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of LPS on plasma ACTH and corticosterone concentrations and pituitary CRF-R1 messenger RNA (mRNA) levels
LPS (50 µg/kg, iv) induced marked and significant (P < 0.01) increases in plasma concentrations of ACTH and corticosterone, as reported previously (Table 1Go).


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  		Table 1. Effect of systemic LPS on plasma ACTH and corticosterone levels

 
RNase protection analysis of total RNA isolated from individual whole pituitaries indicated that 2 h after injection, systemic LPS (50 µg/kg) produced a significant (P < 0.01) decrease in CRF-R1 mRNA levels to 57% of the control value (Fig. 1Go). This decrease in mRNA levels was more marked 6 h after the injection (to 38% of the control value; P < 0.01; Fig. 1Go), but returned toward control levels by 12 and 24 h.



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  		Figure 1. Time course of the effects of LPS on CRF-R1 mRNA levels. Rats were injected iv with saline (control) or LPS (50 µg/kg) and killed 2, 6, 12, and 24 h after injection. Values are the mean ± SEM (group of five or six rats for each treatment). *, P < 0.05; **, P < 0.01 (vs. control). A representative protection assay is shown under the graph. The left portion of the figure shows a representative RNase protection image.

 
Lack of effect of CRF or AVP antiserum or Astressin on the decrease in CRF-R1 after LPS treatment
To determine whether the decrease in CRF-R1 message was primarily due to increased exposure to CRF, LPS was injected concurrently with a CRF antiserum. As shown in Fig. 2Go, the antiserum dramatically reduced the amount of ACTH released in response to LPS alone (from a peak of 1217 ± 213 pg/ml for LPS alone to 51 ± 8 pg/ml for LPS plus anti-CRF, compared to 8 ± 2 pg/ml in controls). Surprisingly, the decrease in CRF-R1 message was not attenuated in the group treated with LPS plus CRF antiserum (Fig. 3Go). Similarly, injection of Astressin, a potent CRF receptor antagonist, did not affect the down-regulation of CRF-R1 mRNA (Fig. 3Go), although it reduced the ACTH response to LPS (Fig. 2Go). As AVP is known to potentiate the effects of CRF on ACTH release and as it has been reported to down-regulate CRF-R1 message in vitro (32), we evaluated whether AVP antiserum administered with LPS could reverse the decrease in CRF-R1 mRNA. However, like CRF antiserum, AVP antiserum did not reverse the diminution in CRF-R1 mRNA levels (Fig. 3Go). Although all treatments dramatically reduced plasma ACTH concentrations, the elevated plasma concentrations of corticosterone produced by LPS (means at 1 h ranged between 23–54 ng/ml in controls and 241–302 ng/ml after LPS) were not significantly affected by anti-CRF antiserum (213 ± 35 ng/ml), Astressin (252 ± 40 ng/ml), or anti-AVP antiserum (289 ± 29 ng/ml), as expected when ACTH responses are not completely abolished (34).



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  		Figure 2. Effect of anti-CRF antiserum (A), Astressin (B), or anti-AVP antiserum (C) on the levels of ACTH after LPS injection. Plasma ACTH levels in vehicle-treated controls and in animals treated with antiserum or antagonist alone were between 5–25 pg/ml (n = 5–6/experimental group).

 


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  		Figure 3. Effect of anti-CRF antiserum (A), Astressin (B), or anti-AVP antiserum (C) on the decrease in CRF-R1 mRNA levels induced by LPS. Rats were killed 6 h after injection. Values are the mean ± SEM (group of five or six rats for each treatment). *, P < 0.05 vs. control. A, Rats were injected with either NSS plus saline (control), NSS plus LPS (LPS, 50 µg/kg), CRF antiserum plus saline (aCRF; 0.15 ml/100 g), or LPS plus CRF antiserum. B, Rats were injected with either saline (control), LPS (50 µg/kg), Astressin (astr; 0.3 mg/ml), or LPS plus Astressin. C, Rats were injected with normal rabbit serum (NRS) plus saline (control), NRS plus LPS (50 µg/kg), AVP antiserum (aAVP, 0.15 ml/100 g) plus saline or AVP antiserum plus LPS.

 
Effect of LPS in vitro
These results prompted us to test in vitro the hypothesis that LPS might have direct effects on pituitary CRF-R1 message levels. Rat anterior pituitary cell cultures were treated with LPS. Treatment with 0.01, 0.1, and 1 µg/ml LPS for 6 h revealed a dose-dependent decrease in CRF-R1 message levels (Fig. 4Go), with no observable change in cell viability. LPS (0.1 µg/ml) for 6 h had no significant effect on ACTH secretion from rat anterior pituitary cells in culture, as reported previously (3).



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  		Figure 4. Effect of LPS on CRF-R1 mRNA levels in rat anterior pituitary cell cultures. Cells were incubated with medium alone (control) or with medium containing 0.01, 0.1, or 1 µg/ml LPS for 6 h. Results are from a representative experiment performed in triplicate. A representative protection assay is shown under the graph.

 
Effect of IL-1ß in vitro
As IL-1ß is known to be induced by LPS in many tissues, including the pituitary (35, 36), and to be the primary mediator of LPS actions, we tested the hypothesis that IL-1 mediates some of the effects of LPS on CRF-R1 mRNA. Cultured rat anterior pituitary cells were incubated for 3 and 6 h in the presence of 1 nM IL-1ß. As shown in Fig. 5Go, IL-1ß treatment for 6 h resulted in a significant decrease in CRF-R1 mRNA levels to 53% of the control value.



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  		Figure 5. Time course of the effect of IL-1ß on CRF-R1 mRNA levels in rat anterior pituitary cell cultures. Cells were incubated with medium alone (control) or with medium containing 1 nM IL-1ß. Results are from a representative experiment performed in triplicate. A representative protection assay is shown under the graph.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we show that systemic inflammation induced by LPS decreases CRF-R1 mRNA expression in the rat pituitary. Inflammatory stress is known to trigger a complex cascade of events to initiate host defense mechanisms that will ultimately tend to restore the organism to homeostasis. Major regulators of this response include the cytokines IL-1, IL-6, and TNF{alpha}, which have been shown to mediate the accompanying effects on the HPA axis (see reviews in Refs. 1–3). For example, systemic IL-1ß rapidly elevates ACTH through stimulation of CRF hypothalamic release (4, 5, 9, 10). Further evidence for an effect of IL-1, IL-6, and TNF{alpha} on hypothalamic CRF release has also been provided by in vitro studies (35, 36, 37). A concomitant increase in the secretion of AVP, another important ACTH secretagogue, has been reported by some investigators (38, 39). Because some studies have reported a decrease in CRF-R1 after stress or adrenalectomy (22, 23), situations that produce elevated CRF secretion, it was expected that after systemic LPS, pituitary exposure to CRF would be the main factor responsible for the decrease in CRF-R1 mRNA levels in the pituitary. In line with this hypothesis, a study with rat anterior pituitary cell cultures revealed that stimulation with CRF can profoundly down-regulate CRF-R1 mRNA levels (32). The ability of AVP to either down-regulate CRF receptors or potentiate the down-regulation induced by CRF in the pituitary has been reported in vivo (40). AVP alone is also sufficient to decrease CRF-R1 mRNA in vitro, although to a lesser extent than CRF (32). Surprisingly, even though it virtually abolished the ACTH response, injection of CRF antiserum before LPS did not prevent the decrease in CRF-R1 mRNA. Similarly, treatment with the CRF receptor antagonist, Astressin, or immunoneutralization against AVP did not attenuate the down-regulation in CRF-R1 mRNA produced by LPS. These findings suggest that LPS may have an effect on CRF receptors that is independent of CRF and AVP, and that either LPS itself or other neuroimmune factors induced by LPS may act on the pituitary to regulate CRF-R1 gene expression.

Indeed, our in vitro data show that in rat pituitary cell cultures, LPS induces a dose-dependent reduction in CRF-R1 mRNA. Although we cannot exclude a direct effect of LPS on corticotropes, LPS is known to trigger the synthesis and release of several immune mediators (1). For example, it is known that the iv administration of endotoxin to laboratory rodents induces marked increases in the plasma concentrations of IL-1ß, IL-6, and TNF{alpha} (41, 42). Moreover, IL-1ß, IL-6, and TNF{alpha} mRNAs have all been demonstrated in the anterior pituitary after peripheral administration of endotoxin (43, 44, 45). Our in vitro data also indicate that IL-1, the primary mediator of LPS actions, reduces the expression of CRF-R1. In agreement with this marked decrease in CRF-R1 mRNA in rat anterior pituitary cells, IL-1ß was recently reported to decrease CRF receptor mRNA in the rat pituitary after systemic injection (46). In our rat anterior pituitary cell studies, the decrease in CRF-R1 mRNA was already significant after 3-h incubation with IL-1, suggesting that this inflammatory mediator could be responsible, at least partly, for the decrease in CRF-R1 that we noted in vivo in response to LPS. The present evidence suggests that in contrast to the mouse corticotrope cell line, AtT-20 (47), normal mouse corticotropes do not express IL-1 receptors (48), suggesting that the effects of IL-1 that we observed were not due to direct actions on corticotropes, but are mediated via interactions with other pituitary cell types. Although we found that IL-1 reduces CRF-R1 mRNA in vitro, we cannot exclude that other inflammatory mediators, such as IL-6 and TNF{alpha}, known to be increased in conditions linked to the activation of the immune system, might also participate in the down-regulation of CRF-R1 mRNA due to LPS. During infection or inflammation, these cytokines are produced in the systemic circulation as well as in tissues of the HPA axis, including the pituitary (2, 43, 44, 45). Interestingly, during local inflammation induced by turpentine in the rat, which produces high circulating levels of IL-6, we also observed a significant decrease in CRF-R1 in the pituitary (Turnbull, A. V., J. M. Aubry, G. Pozzoli, C. Rivier, and W. Vale, unpublished observations).

Glucocorticoids also modulate pituitary CRF receptor expression. In intact rats, chronic administration of corticosterone (49) causes a dose-dependent decrease in CRF-binding sites of the anterior pituitary. Similarly, in vitro, a diminution of CRF receptor level and CRF-R mRNA has been found in cultured pituitary treated with glucocorticoids (32, 50). In the AtT-20 cell line, prolonged incubation with dexamethasone reduced CRF binding by 80% (51). In our study, even though the CRF/AVP antisera and CRF receptor antagonist significantly reduced ACTH secretion during LPS treatment, plasma corticosterone levels remained high compared to those in control animals. Therefore, we cannot exclude the possibility that corticosterone may contribute to the reduction in corticotropic CRF receptors during endotoxemia.

In conclusion, we have shown that the rat pituitary CRF-R1 mRNA is reduced by systemic LPS treatment, through mechanisms that appear independent of increases in CRF/AVP levels. Our results indicate that LPS itself or inflammatory mediators induced by LPS (e.g. IL-1) may act at the pituitary level and be at least partially responsible for the decrease in CRF receptor, which illustrates the complex regulatory pathways that control CRF-R1 expression in response to immune challenge.


   Acknowledgments
 
The authors thank Dr. J. Rivier for generously providing synthetic peptides and the CRF receptor antagonist Astressin, and R. Kaiser for their synthesis. We also acknowledge A. Corrigan and C. Donaldson for technical assistance, and L. Bilezikjian for helpful discussions.


   Footnotes
 
1 This work was supported by the Swiss Fond National for Scientific Research (to J.-M.A.), Amoco and Aaron Fellowships (to A.V.T.), Universita Cattolic del Sacro Cuore (to G.P.), NIH Grant DK-26741 (to C.R. and W.V.), and the Foundation for Research (to W.V. and C.R.). Back

2 Investigator with the Foundation for Research. Back

3 Senior Investigator with the Foundation for Research. Back

Received September 9, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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